Objective for evanescent illumination and microscope

ABSTRACT

A microscope comprises an objective and a light source that produces an illumination light beam—in particular for evanescent illumination of a sample, which exhibits a focus in the plane of the objective pupil. To adjust the penetration depth, an adjustment mechanism is provided with which the spatial position of the focus within the plane of the objective pupil may be changed.

RELATED APPLICATIONS

This application is a Continuation of PCT application serial numberPCT/EP04/52287 filed on Sep. 23, 2004 which in turn claims priority toGerman application serial number DE 103 44 410.6 filed on Sep. 25, 2003and German application serial number DE 10 2004 044 308.4 filed on Sep.10, 2004, both of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to a microscope with an objective and with a lightsource that produces an illumination light beam—in particular forevanescent illumination of a sample, which exhibits a focus in the planeof the objective.

The invention further relates to an illumination module with a lightsource that produces an illumination beam.

BACKGROUND OF THE INVENTION

The microscope with evanescent illumination of a sample is known from US2002/0097489 A1. The microscope comprises a white light source, thelight of which is coupled for the purpose of evanescent illumination viaa slit aperture through the microscope objective onto a sample holder,which holds a sample. The illumination light propagates itself in thesample holder by means of total internal reflection, whereby theillumination of the sample occurs only in the region of the evanescentfield that protrudes from the sample holder. Microscopes of this typeare known as “total internal reflection fluorescent microscopes”(TIRFM).

The z-resolution of TIRF microscopes is extraordinarily good because theevanescent field protrudes only about 100 nm into the sample.

A high-aperture objective specifically for TIRF application is knownfrom DE 101 08 796 A1. The objective comprises a first lens withpositive refractive power and a second lens with negative refractivepower, whereby the focal distance ratio between the two lenses is in the−0.4 and −0.1 range, and the total refractive power is greater thanzero. The objective further comprises two positive lenses, the diameterratio to focal length of which is greater than 0.3 and less than 0.6.The objective further comprises a negative lens and a collecting lens,whereby the negative lens faces the front group, and the focal distanceratio of the negative lens to the collector lens is between −0.5 and −2.

An incident illumination device for TIRF microscopy is known from DE 10217 098 A1. The incident illumination device comprises an illuminationsource that emits a polarized illumination beam when in operation, whichpropagates at an angle to the optical axis and a deflector that deflectsthe illumination light beam and couples it parallel to the optical axisin the objective. Provision is made in this incident illumination devicefor the illumination light beam emitted by the illumination source toexhibit a phase difference in the s- and p-polarization directions, andfor the deflection arrangement to reflect the illumination light beam xtimes, whereby x=(n×180°−d)/60°.

A microscope for total internal reflection microscopy (TIRM) is knownfrom DE 101 43 481 A1. The microscope exhibits a microscope housing andan objective. The illumination light emitted by an illumination devicecan be coupled via an adapter that can be inserted into the microscopehousing.

A microscope with an optical illumination system that enables simpleswitching between evanescent illumination and reflective illumination isknown from US 2004/0001253 A1. The illumination system comprises a laserlight source, the light of which is coupled in an optical fiber.Furthermore, an outcoupling optic is provided that focuses the outgoinglight that from the fiber onto a rear focal point of the microscopeobjective. The optical fiber is movable along a plane that isperpendicular to the optical axis of the microscope objective.

A device for coupling light in a microscope is known from DE 102 29 935A1. Here, a laser light is directed onto a sample in the illuminatedfield diaphragm plane by a laser light fiber coupling, which isimplemented as a slide. The invention is particularly suitable for theTIRF method.

In scanning microscopy, a sample is illuminated with a light beam toobserve the detection light emitted by the sample as reflection orfluorescent light. The focus of an illumination light beam is moved onan object plane with the help of a movable beam deflector, generally bytipping two mirrors, whereby the axes of deflection are usuallypositioned perpendicular to each other, so that one mirror deflects inthe x-direction and the other in the y-direction. The mirrors are tippedwith the help of galvanometric positioners, for example. The power ofthe light coming from the object is measured dependent on the positionof the scanning beam. Generally, the positioners are provided withsensors to determine the actual position of the mirrors. In confocalscanning microscopy, in particular, an object is scanned in threedimensions with the focus of a light beam.

A confocal scanning microscope generally comprises a light source, afocusing optic with which the light from the source is focused on apinhole aperture—the so-called excitation aperture, a beam splitter, abeam deflector to control the beam, a microscope optic, a detectionaperture, and detectors to detect the detection light or fluorescentlight. The illumination light is coupled via a beam splitter. Thefluorescent light or reflected light emitted by the object returns tothe beam splitter via the beam deflector, passes through it, and issubsequently focused onto the detection aperture, behind which arelocated the detectors. This arrangement of detectors is called a descanarrangement. Detection light that does not originate directly from thefocal region takes another light path and does not pass through thedetection aperture so that pixel information is obtained, which isconverted into a three-dimensional image by sequential scanning of theobject with the focus of the illumination light beam. A 3-dimensionalimage is usually achieved by means of layered image data.

SUMMARY OF THE INVENTION

It is a task of the present invention to disclose a microscope thatenables variable adjustment of the penetration depth of illuminationlight, in particular for evanescent illumination of a sample.

This task is solved by a microscope, wherein an adjustment mechanism isprovided with which the spatial position of the focus within the planeof the objective pupil may be changed.

A further task of the present invention is to disclose an illuminationmodule for a microscope that enables illumination of a microscopicsample, in particular for evanescent sample illumination with adjustablepenetration depth.

The further task is solved by an illumination module, wherein theillumination module may be coupled to a microscope such that theillumination light beam in the plane of the objective pupil of themicroscope exhibits a focus, and wherein the illumination modulecomprises an adjustment mechanism with which the spatial position of thefocus within the plane of the object pupil may be changed.

It has been recognized, according to the invention, that the penetrationdepth of an evanescent illumination field in a sample is dependent onthe angle at which total reflection at the cover glass interface or atthe sample holder interface occurs. This angle is directly correlatedwith the angle relative to the optical axis at which the illuminationlight beam which is provided for evanescent sample illumination exitsfrom the objective via the front lens. This angle, in turn, is dependentupon the distance from the optical axis at which the illumination lightbeam passes through the rear focal plane of the objective (pupil). Inorder to have available a largely parallel illumination light beam forthe purpose of evanescent sample illumination, the illumination lightbeam must exhibit a focus in the rear focal plane of the objective.Finally, the distance of the focus to the optical axis of the objectivedetermines the aforementioned angle, and therewith the penetration depthof the evanescent field in the sample to be tested.

In a preferred embodiment of the microscope according to the invention,the adjustment mechanism comprises an adjustable beam deflector that isarranged in the beam path of the illumination light beam. Preferably,the beam deflector comprises at least one galvanometric mirror. In orderto position the focus at any given location within the objective pupil,the beam deflector preferably comprises two galvanometric mirrors, whichcause deflection of the illumination light beam in different lateraldirections (e.g., x- and y-direction). The beam deflector may alsocomprise rotatable or tippable prisms and/or rotatable or tippablemirrors. The use of acousto-optical or electro-optical deflectionelements can also be envisioned.

In a further embodiment of the invention, the adjustment mechanismcomprises a light-conducting fiber which is at least partially movable.In this variant, mechanical positioners are preferably provided thatenable the light outgoing end of the light-conducting fiber to bepositioned precisely within the objective pupil. The illumination lightbeam in this further development of the invention is focused onto thelight incoming end of the light-conducting fiber, conveyed through thelight-conducting fiber, and de facto again exhibits a focus at theoutcoupling end that is positioned within the objective pupil, becauseof the small diameter of customarily used light-conducting fibers.

As previously explained, it is particularly important to adjust thedistance of the focus of the illumination light bean in the objectivepupil relative to the optical axis of the objective in order to adjustthe penetration depth of the evanescent field in the sample region.

It can be particularly advantageous for certain applications to drivethe adjustment mechanism such that the focus describes a selectablecurve path within the objective pupil plane. By so doing, particularlyhomogeneous illumination can, for example, be achieved. In certainexperiments, it is possible with this variant to effect constantalternation in polarization direction. In a particularly preferredembodiment of the invention, the curve path is a circular path. Anembodiment of the invention in which the curve path is a circular paththe midpoint of which lies on the optical axis of the objective is veryparticularly preferred. In this variant, the penetration depth remainsconstant while the focus describes the circle of the curve path,whereby, however the illumination light beam that exits from theobjective is continuously coupled to the cover glass or to the sampleholder from various directions. It is also possible to select differentcoupling directions in order to compare the resultant, possiblydifferent, images of the sample.

In a very preferred variant, a compensating optic is provided in orderto compensate for unevennesses in the objective pupil plane.

Preferably, the microscope objective is exchangeable (e.g., objectiveturret), whereby a compensating optic is provided to compensate for thevarious pupil positions of different objectives. The distances betweenthe front focal plane and the rear focal plane may differ from objectiveto objective, which may lead to problems because in order to achieveoptimal evanescent sample illumination, the focus of the illuminationlight beam must lie more or less exactly in the objective pupil. Theaforementioned compensating optic, which may, for example, be a zoomoptic or several exchangeable optics arranged on a turret, compensatesfor these differences in distance.

In a preferred embodiment of the microscope according to the invention,a light trap is provided to eliminate unused illumination light. Only aportion of the illumination light coupled to the cover glass or sampleholder actually evanescently illuminates the sample. The light, whichagain exits from the cover glass or from the sample holder after severaltotal reflections, many return to the microscope again and lead toimaging disturbances (as a result, for example, of scattered light).This is avoided, according to the invention, by a suitably arrangedlight trap.

In a particularly preferred embodiment of the microscope, a switch isprovided for switching between classic incident illumination andevanescent sample illumination. The switch may, for example, comprise awing mirror.

Preferably, the light cone is variable, in particular for changing theazimuth. An aperture optic such as an iris optic arranged in theintermediate image plane may be provided in order to set the diameter ofthe illumination light beam that exits from the microscope objective.

In a particular embodiment a camera is provided for imaging. The cameramay, in a particularly preferred variant, be implemented as a colorcamera, or as a CCD camera.

Preferably, the objective exhibits a numeric aperture that is greaterthan 1.4, in particular greater than 1.45, in particular greater than1.6. Preferably, the numeric aperture of the objective is 1.45 or 1.65.

In a particular variant, at least the light source and the adjustmentmechanism are incorporated in a single illumination module, which canpreferably be coupled to a microscope and/or a microscope stand.

The illumination module, according to the invention, offers theadvantage that it may be coupled as a retrofit to a preexistentmicroscope or microscope stand.

BRIEF DESCRIPTION OF THE DRAWINGS

The object of the invention is schematically represented in the diagram,and is described below on the basis of figures, wherein elements thathave the same function are given the same reference numbers. They show:

FIG. 1 a microscope according to the invention;

FIG. 2 a further microscope according to the invention:

FIG. 3 an illumination module that is coupled to a microscope;

FIG. 4 a microscope with an illumination module; and

FIG. 5 a further microscope according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 shows a microscope 1 according to the invention, with an opticaldevice 3 for manipulating a sample 5. The optical device 3 comprises alight source 7 that emits a manipulation light beam 9, and an adjustablebeam deflector 11. The microscope 1 comprises an objective 13 and afurther light source 15 that is implemented as a laser 17, and whichproduces an illumination light beam 19. The illumination light beam 19that is emitted from the further light source 15, serves to evanescentlyilluminate the sample 5, which is positioned on a sample holder 21. Inorder to achieve evanescent sample illumination, the wing mirror 23 istipped in the position indicated by the broken lines. By so doing, thebeam path is cleared for the illumination light beam 19. A compensatingoptic 25 is provided in the beam path of the illumination light beam 19to compensate for unevennesses in the objective pupil plane 27. Thecompensating optic 25 also enables compensation for different pupilpositions of different objectives 13. For this purpose, the compensatingoptic 25 is implemented so as to be axially movable. The illuminationlight beam 19 exhibits a focus 31, which is indicated by a point, in theplane 27 of the objective pupil 29, and which may be varied in itsposition in the plane 27 of the objective pupil 29 with the help of thefurther adjustable beam deflector 32.

Several optical elements for directing and forming the beam are arrangedin the beam path of the microscope 1 There may, for example, be a firstoptic 33, a second optic 35, and an optic 25, which produce a firstintermediate image plane 37 and a second intermediate image plane 39.The adjustable further beam deflector 32 comprises a cardanicallysuspended rotating mirror 41, which is not shown. The distance of thefocus 31 to the optical axis 43 of the objective 3 may be adjusted andtherewith the penetration depth of the illumination light beam in thesample 5 varied with the help of the further adjustable beam deflector31. The detection light 45 exiting from the sample 5 passes through theobjective 3 as well as through the beam splitter 47, which directs theillumination light beam 19 through the objective 3 and through the tubeoptic to a detector 49, which is implemented as a CCD camera 51. Thebeam splitter 47 is implemented as a dichroic beam splitter, and isdesigned such that the light of the wavelength of the illumination lightbeam 19 is reflected, whereas light of the wavelength of the detectionlight 47 may pass through.

FIG. 2 shows a variant in which the optical device 3 for manipulating asample 5 produces both a manipulation light beam 9 and an illuminationlight beam 19. The device 3 for manipulating a sample 5 comprises alight source 7, a multi-linear laser 53, from the emission light ofwhich the portions of the desired wavelengths are selectable with anAOTF 55. The compensating optic 57 is introduced into the beam path ofthe microscope 1 for the purpose of evanescently illuminating the sample5. The compensating optic 57 enables the illumination light beam 19 toexhibit a focus 31, which is indicated by a point, in the plane 27 ofthe objective pupil 29. For the purpose of sample manipulation, thecompensating optic 57 is removed from the beam path so that themanipulation light beam 9 is focused on the sample. Both the penetrationdepth of the illumination light in the sample (by adjusting the positionof the focus of the illumination light beam 19 in the plane 27 of theobjective pupil 29) and sample manipulation are controlled with the beamdeflector 11.

FIG. 3 shows a variant of a microscope 1 according to the invention, inwhich the illumination light beam 19 passes through the compensatingoptic 25, which makes it possible for the illumination light beam 19 toexhibit a focus 31, which is indicated by a point, in the plane 27 ofthe objective pupil 29, while the manipulation light beam 9 is directedat the compensating optic 25 via the first deflecting mirror 59, thesecond deflecting mirror 61, the third deflecting mirror 63, and thefourth deflecting mirror 65.

FIG. 4 shows a variant of the microscope shown in FIG. 1. In thisvariant, the optical device 3 for manipulating a sample 5 comprises anillumination pinhole aperture 67, a detection pinhole aperture 69, whichis arranged before a multi-band detector 71, as well as a dichroic beamsplitter 77 for deflecting the further detection light 73, which issuesfrom the sample, into the detection beam path 75. The microscope shownenables confocal observation of the sample 5, whereby confocalillumination is achieved with the focus of the manipulation light beam9.

The invention was described in relation to a particular embodiment.However, it is clear that changes and variations may be implementedwithout abandoning the scope of the following claims.

1. Microscope with an objective and with a light source that produces anillumination light beam—in particular for evanescent illumination of asample, which exhibits a focus in the plane of the objective pupil,wherein an adjustment mechanism is provided with which the spatialposition of the focus may be changed within the plane of the objectivepupil.
 2. Microscope according to claim 1, wherein the adjustmentmechanism comprises an adjustable beam deflector, which is arranged inthe beam path of the illumination light beam.
 3. Microscope according toclaim 2, wherein the beam deflector comprises at least one galvanometricmirror.
 4. Microscope according to claim 1, wherein the adjustmentmechanism comprises a light-conducting fiber, which is at leastpartially movable.
 5. Microscope according to claim 1, wherein adistance of the focus of the illumination light beam to the optical axisof the objective is adjustable.
 6. Microscope according to claim 1,wherein the adjustment mechanism is controllable such that the focusdescribes a selectable curve path within the plane of the objectivepupil.
 7. Microscope according to claim 6, wherein the curve path is acircular path.
 8. Microscope according to claim 1, wherein acompensating optic is provided to compensate for unevennesses in theobjective pupil plane.
 9. Microscope according to claim 1, wherein themicroscope objective is exchangeable, and wherein a compensating opticis provided to compensate for different pupil positions.
 10. Microscopeaccording to claim 1, wherein a light trap is provided to eliminateunused illumination light.
 11. Microscope according to claim 1, whereina switching mechanism is provided to switch between incidentillumination and evanescent illumination.
 12. Microscope according toclaim 11, wherein the switching mechanism comprises a wing mirror. 13.Microscope according to claim 1, wherein the light cone is variable, inparticular to change the azimuth.
 14. Microscope according to claim 1,wherein a camera is provided for imaging.
 15. Microscope according toclaim 1, wherein the objective exhibits a numeric aperture that isgreater than 1.4, in particular greater than 1.45, in particular greaterthan 1.6, and that is preferably 1.45 or 1.65.
 16. Microscope accordingto claim 1, wherein at least the light source and the adjustmentmechanism are incorporated in an illumination module.
 17. Microscopeaccording to claim 16, wherein the microscope exhibits a microscopestand, and wherein the illumination module is detachably coupled to themicroscope stand.
 18. Illumination module with a light source thatproduces an illumination light beam, wherein the illumination module maybe coupled to a microscope such that the illumination light beamexhibits a focus in the plane of the objective pupil of the microscope,and wherein the illumination module incorporates an adjustment mechanismwith which the spatial position of the focus may be changed within theplane of the objective pupil.
 19. Illumination module according to claim18, wherein the adjustment mechanism comprises an adjustable beamdeflector arranged within the beam path of the illumination light beam.20. Illumination module according to claim 19, wherein the beamdeflector comprises at least one galvanometric mirror.
 21. Illuminationmodule according to claim 18, wherein the adjustment mechanism comprisesa light-conducting fiber that is at least partially movable. 22.Illumination module according to claim 18, wherein a distance of thefocus of the illumination light beam to the optical axis of theobjective may be adjusted.
 23. Illumination module according to to claim18, wherein the adjustment mechanism is controlled such that the focuscontinually describes a selectable curve path within the plane of theobjective pupil.
 24. Illumination module according to claim 23, whereinthe curve path is a circular path.
 25. Illumination module according toclaim 18, wherein a compensating optic is provided to compensate forunevennesses in the objective pupil plane.
 26. Illumination moduleaccording to claim 18, wherein the microscope stand is exchangeable, andwherein a compensating optic is provided to compensate for differentpupil positions.
 27. Illumination module according to claim 18, whereina light trap is provided to eliminate unused illumination light. 28.Illumination module according to claim 18, wherein a switching mechanismis provided to switch between incident illumination and evanescentillumination.
 29. Illumination module according to claim 28, wherein theswitching mechanism comprises a wing mirror.
 30. Illumination moduleaccording to claim 18, wherein the light cone is variable, in particularto change the azimuth.